Comparison of mouse ovarian follicular development and gene expression in the presence of ovarian tissue extract and sodium selenite: An experimental study

Abstract Background Ovarian tissue extract (OTE) and sodium selenite (SS) enhance the growth and maturation of preantral follicles in a dose-dependent manner. Objective The present study was designed to bring more information regarding the mechanism of OTE and SS on the mRNA expression of follicle-stimulating hormone receptors (FSHR) and the proliferation cell nuclear antigens (PCNA) of in vitro matured isolated follicles. Materials and Methods The tissue extract was prepared from adult ovaries. The preantral follicles (n = 266) were isolated from 12-16-day-old mice and cultured in the control, experimental I (10 ng/ml SS), and experimental II (OTE) groups for 12 days. The follicular diameter, survival, and maturation rates, also, the production of 17-β-estradiol and progesterone, and the follicular expression of PCNA and FSH receptor genes were analyzed. Results The survival rate of follicles in the SS-treated group (84.58%) was significantly higher than that OTE (75.63%; p = 0.023) and control (69.38%; p = 0.032) groups. The mean diameter of culture follicles in experimental group I (403.8 μm) and experimental group II (383.97 μm) increased significantly in comparison with the control group (342.05 μm; p = 0.032). The developmental rate of follicles, percentages of antrum formation, released metaphase II oocytes (p = 0.027; p = 0.019 respectively), production of hormones and the expression of 2 studied genes were significantly increased in both experimental groups in compare with control group (p = 0.021; p = 0.023 respectively). Conclusion The OTE and SS have a positive effect on development of mouse preantral follicles via over-expression of FSHR and PCNA genes.


Introduction
Folliculogenesis in mammals is one of the most complex processes that involves direct interaction between somatic cells (such as granulosa and theca cells) and oocytes (1). Different factors such as hormones, growth factors, and antioxidants play critical roles in the growth and maturation of follicles and oocytes (2). Several culture systems, including 2-and 3-dimensional systems, were designed to investigate the effects of these factors during in vitro follicular culture. Culture media usually contain necessary supplementations such as serum, growth factors, hormones, and so on that improve follicular maturation (3).
It has been reported that selenium salts as non-metal chemical elements in large amounts are very toxic, but trace amounts of selenium are necessary for animal cell function (4). The addition of selenium to the culture medium as an antioxidant leads to the maturation of follicles and oocytes (5)(6)(7)(8)(9)(10). It was reported that the supplementation of culture media with sodium selenite (SS) could increase the growth and maturation of preantral follicles (5). Also, several studies have demonstrated that supplementation of mouse oocyte maturation media with SS improved not only the oocyte developmental rate but also enhanced its expression of mitochondrial transcription factor A and mitochondrial DNA (6)(7)(8)(9). These studies have shown that the reactive oxygen species (ROS) level was decreased in the presence of SS, which acts as an antioxidant (5,7). It was also shown that selenium is one of the main critical factors in follicular fluid (11).
Ovarian follicular development is also regulated by intra-ovarian factors such as steroid hormones, epidermal growth factor, vascular growth factor, fibroblast growth factor, and growth differentiation factor β that enhance follicular development (11,12). Some of these factors are produced by the ovarian stromal cells and follicular cells (12). The ovarian microenvironment could improve follicular development by providing these elements. However, the literature shows that supplementation of culture media with follicular fluid could enhance the development and the quality of cumulus-oocyte complex and embryo (12,13).
Previously, we studied the effects of different concentrations of ovarian tissue extract (OTE) on mouse follicular development. The OTE contains components that mimic the natural composition of ovarian tissues and was obtained from adult mouse ovaries, which contain a lot of follicular fluid that has an impact on follicular development. Our results at the morphological level demonstrated that the OTE increased the development of follicles as its concentration increased within the culture medium (14).
Moreover, this study was designed to bring some additional information and compare the effect of SS and OTE on follicular function and development.
In addition, at the mRNA level, the expression of 2 critical genes including follicle-stimulating hormone receptor (FSHR) and proliferation cell nuclear antigen (PCNA) that are involved in the maturation and proliferation of were assessed.

Materials and Methods
The reagents were prepared by Sigma Aldrich company (Dusseldorf, Germany) except those indicated in the text.

Study design
In this experimental study from mouse ovaries, the follicles were dissected with 145-155 μm diameter and divided into 3 groups as follows. In the control group the culture media (α-minimum essential medium; αMEM) containing 1% insulin, transferrin, and selenium (Invitrogen, Paisley, UK), 100 mIU/ml recombinant follicle-stimulating hormone (rFSH or Gonal-f; Serono, Switzerland), 100 IU/ml penicillin and 100 mg/ml streptomycin and 10% fetal calf serum (FBS; Gibco, UK). In experimental group I, the culture media was the same as the control, in addition, 10 ng/ml SS was added (5). In experimental group II, the described culture media was used and the FBS was replaced with OTE (14). After 12 days of culture, the survival and developmental rate of follicles and the expression of FSHR and PCNA genes were analyzed and compared. In addition, the levels of 17-β-estradiol and progesterone in the collected media were analyzed.

Animals and collection of ovarian tissue
For this study, 12-16-day-old female NMRI mice (n = 20) ovaries that contain preantral follicles were used for follicular isolation and 6-8-wkold adult mice (n = 15) for the preparation of OTE. The mice were maintained in the animal house of the university under standard conditions (12-hr light/12-hr dark cycle, 22-24°C, and 55% humidity). The ovaries were obtained after mice cervical dislocation, then transferred to culture media consisting of 10% FBS, 100 IU/ml penicillin, and 100 mg/ml streptomycin until assessments.

OTE preparation
The OTE was prepared according to the previous study (14). After dissecting the adult mice ovaries under a stereomicroscope, they (n = 15) were collected and immersed in potassium ammonium chloride solution (pH = 7.2-7.4) for 1 min. Ovaries were then washed 3 times with phosphate-buffered saline (PBS) and put into a glass homogenizer tube. They were homogenized by adding 2 ml of Tris-HCL buffer (pH = 7) and 50 μL of protease inhibitor solution. Then the volume of the solution reached 10 ml after the homogenization of ovarian tissues by Tris-HCL buffer (pH = 7). The obtained solution was sonicated for 2 min under W50 sonication conditions. Finally, the solution was centrifuged at 12000 RPM for 15 min. The supernatant was collected as tissue extract and stored at -80°C until assay.

Total protein analysis
500 μL of supernatant of OTE was used to measure the amount of protein. The amount of protein in fetal bovine serum (FBS) was used as a reference to select the concentration of OTE. According to our previous study, the OTE which contains ½ of the protein level in FBS was selected (14).

3-dimensional follicle culture
A 3-dimensional culture system based on sodium alginate capsulation was used for follicular culture (14). A mixture of sodium alginate and activated charcoal in distilled water was prepared to eliminate organic pollution. After 24 hr it was diluted with PBS at a concentration of 0.5% (w/v), then each isolated follicle was individually transferred into 7 µL of sodium alginate and a cross-linking solution containing 50 mM CaCl 2 and 140 mM NaCl, respectively. After washing the follicles in PBS at least 2 times for 2 min in each, the capsulated follicles (n = 248 in total) were put in the medium under mineral oil. The group without SS and OTE was considered as the control group (n = 90) and in this group, the αMEM culture media was supplemented with 1% insulin, transferrin, and selenium, 100 mIU/ml rFSH, 100 IU/ml penicillin, and 100 mg/ml streptomycin and 10% f FBS. In experimental group I (n = 86 follicles) the FBS was replaced by 10 ng/ml SS (5) and in experimental group II (n = 90 follicles) with OTE as its protein concentration was ½ of FBS protein (14). The half of culture media was renewed by fresh ones at one-day intervals during the culture period and the remaining media was frozen until analysis.

In vitro ovulation induction
The morphological changes of follicles were studied using inverted microscope, and the follicles that showed a dark appearance were considered dead. By supplementing the culture media with 1.5 IU/ml of human chorionic gonadotropin (hCG; Organon) the oocyte ovulation induction was induced on Day 12 of culture, then 18 hr later the oocyte staging was done. The germinal vesicle oocyte has the prominent nucleus that is arrested in the prophase of meiosis I; the nucleus and polar body were not visible in the germinal vesicle breakdown oocyte, and the presence of the first polar body was demonstrated in the metaphase II (MII) oocyte (13).

Hormonal assays
At the end of the culture period, steroid hormones, including 17-β-estradiol and progesterone, were analyzed in the collected media (n = 3 in each group). The levels of 17-β-estradiol and progesterone (Biotest AG, Dreieich, Germany) were measured by an enzyme immunoassay, and these experiments were done in triplicates.

RNA extraction and cDNA synthesis for molecular assessment
Total RNA was extracted from follicles in each studied group (n = 12 in 3 replicates) using a TRIzol reagent extraction method (Invitrogen, Paisley, UK). By spectrophotometer, the quality of extracted RNA and its level were analyzed. The list of applied primers for PCNA, FSHR, and -actin was summarized in table I.

Real-time reverse transcription polymerase chain reaction (RT-PCR)
After extraction of total RNA and cDNA synthesis, according to QuantiTect SYBR Green RT-PCR kit (Applied Biosystems, UK), real-time RT-PCR was performed. Thermal conditions for the process included 3 steps; holding stage (95ºC for 5 min), step 2 was performed at 95ºC for 15 s, 58ºC for 30 s, and 72ºC for 30 s, and the last step (melt curve step) was continued at 95ºC for 15 s, 60ºC for 1 min, and 95ºC for 15 s. Then, the relative quantification of target genes to housekeeping genes was determined.

The diameter of cultured isolated preantral follicles
The

Follicular developmental rate
The survival and antrum formation rates in follicles and the rate of released MII oocytes are summarized in table III. At the end of the culture period, amongst the studied follicles in control, SS, and OTE-supplemented groups, the survival rates were found to be higher in SS supplemented group (p = 0.032). In the control group the percentages of antrum formation and the rate of released MII oocytes were significantly lower than in other studied groups (p = 0.027); but there was no statistical difference between SS and OTE groups.

Hormonal assay
The levels of estradiol and progesterone in the group that was treated with SS and those treated with OTE were increased (p = 0.021) but there was no significant difference between these groups in this regard (Table IV).

The expression of PCNA and FSHR genes
The mRNA levels of PCNA and FSHR genes were evaluated by real-time RT-PCR. The expression ratio of these target genes    Data presented as Mean ± SD. One-way ANOVA and Tukey's HSD. (*): Shows significant differences with the control group in the same column (p = 0.021). There was no significant difference between experimental groups I and II. SS: Sodium selenite, OTE: Ovarian tissue extract

Discussion
The present study aimed to evaluate the FSHR is necessary for follicular development and is also responsible for producing androgens (25).
It has been reported that when the transition from the primordial to primary follicle occurred, FSHR expresses progressively (26). The presence of FSHR in the early follicular stages has an indirect effect on follicular development via paracrine factors released by ovarian stromal cells (24,27).

Conclusion
The OTE and SS have a positive effect on in vitro growth and maturation of mouse preantral follicles via over-expression of FSHR and PCNA genes.